U.S. patent application number 12/754533 was filed with the patent office on 2010-10-07 for optical detection alignment coupling apparatus.
Invention is credited to Varouj Amirkhanian, Paul Mooney.
Application Number | 20100252437 12/754533 |
Document ID | / |
Family ID | 46304713 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100252437 |
Kind Code |
A1 |
Amirkhanian; Varouj ; et
al. |
October 7, 2010 |
OPTICAL DETECTION ALIGNMENT COUPLING APPARATUS
Abstract
An apparatus for aligning a capillary column with one or more
excitation fibers and with one or more optical lens elements for
Capillary Electrophoresis. The apparatus includes two identical
blocks having a plurality of grooves for positioning and aligning
the capillary column with the one or more excitation fibers, and a
plurality of lens seats for optically coupling the lens element
with the capillary column. Each block includes a male and female
part for mating the two identical blocks together.
Inventors: |
Amirkhanian; Varouj; (La
Crescenta, CA) ; Mooney; Paul; (Rancho Santa
Margarita, CA) |
Correspondence
Address: |
LIU & LIU
444 S. FLOWER STREET SUITE 1750
LOS ANGELES
CA
90071
US
|
Family ID: |
46304713 |
Appl. No.: |
12/754533 |
Filed: |
April 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11152030 |
Jun 13, 2005 |
7691247 |
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12754533 |
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PCT/US03/39971 |
Dec 15, 2003 |
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11152030 |
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10319803 |
Dec 13, 2002 |
7250099 |
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PCT/US03/39971 |
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Current U.S.
Class: |
204/602 |
Current CPC
Class: |
G01N 2021/6463 20130101;
G01N 21/645 20130101; G01N 2021/6484 20130101; G01N 27/44721
20130101 |
Class at
Publication: |
204/602 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Claims
1. An apparatus for aligning a capillary, for supporting a sample,
with respect to a sample analysis system including a fiber, the
apparatus comprising: an alignment block having an outer face and
an inner face; a plurality of grooves defined on the outer face,
wherein the plurality of grooves intersect about at a detection
point on the alignment block, wherein each of the plurality of
grooves is sized and shaped to nest one of either the capillary or
the fiber; and a support block engaged with the outer face for
maintaining nesting the capillary and the fiber within the
plurality of grooves.
2-26. (canceled)
Description
[0001] This is a Continuation of International Application
PCT/US03/39971, with an international filing date of Dec. 15, 2003,
which is a Continuation-in-Part of U.S. patent application Ser. No.
10/319,803, filed Dec. 13, 2002; these applications are fully
incorporated by reference as if fully set forth herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to bio-separation systems, and
more particularly a coupler for aligning optical detection
components.
[0004] 2. Description of Related Art
[0005] Bioanalysis, such as DNA analysis, is rapidly making the
transition from a purely scientific quest for accuracy to a routine
procedure with increased, proven dependability. Medical
researchers, pharmacologists, and forensic investigators all use
DNA analysis in the pursuit of their tasks. Yet due to the
complexity of the equipment that detects and measures DNA samples
and the difficulty in preparing the samples, the existing DNA
analysis procedures are often time-consuming and expensive. It is
therefore desirable to reduce the size, number of parts, and cost
of equipment, to ease sample handling during the process, and in
general, to have a simplified, low cost, high sensitivity
detector.
[0006] One type of DNA analysis instrument separates DNA molecules
by relying on electrophoresis. The term electrophoresis refers to
the movement of a charged molecule under the influence of an
electric field. Electrophoresis can be used to separate molecules
that have equivalent charge-to-mass ratios but different masses.
DNA fragments are one example of such molecules. Electrophoresis
techniques could be used to separate fragments of DNA for
genotyping applications, including human identity testing,
expression analysis, pathogen detection, mutation detection, and
pharmacogenetics studies.
[0007] There are a variety of commercially available instruments
applying electrophoresis to analyze DNA. One such type is a
capillary electrophoresis (CE) instrument. CE instruments employ a
fused silica capillary column carrying a buffer solution. A DNA
sample is capable of being introduced through the capillary column
by electrophoresis. When electrophoresis is applied to the
capillary column, the DNA sample separates into its components, and
the components migrate through the capillary column to a detection
window where the DNA components can be analyzed.
[0008] There are detection techniques well known in the art for
analyzing the DNA components. Radiation absorption detection is one
such well-known technique that involves directing incident
radiation at the analytes in the detection window and measuring the
amount or intensity of radiation that passes through the analytes,
or the equivalent decrease in intensity or the amount of radiation
that is absorbed by the analytes (i.e., the attenuation of the
incident radiation).
[0009] Another well-known detection technique is Emissive radiation
detection. Fluorescence detection, such as Laser-induced
fluorescence (LIF) detection methods, is often the detection method
of choice in the fields of genomics and proteomics because of its
outstanding sensitivity compared to other detection methods. The
DNA sample is tagged with a fluorescent material. The DNA
components can be analyzed by directing light through the capillary
wall at the detection window, at the tagged components, and
detecting the fluorescence emissions induced by the incident light.
The intensities of the emission are representative of the
concentration, amount and/or size of the components of the
sample.
[0010] There are numerous challenges in designing CE-based
instruments and CE analysis protocols. To maximize signal intensity
and sensitivity and resolution of detection, the precise position
and alignment of particular CE instrument components, such as the
capillary column, the excitation light fiber and the detection
lens, with respect to each other are critical design concerns. The
capillaries used in CE are relatively small, ranging in size from
20 .mu.m to 250 .mu.m I.D., and CE requires that the detection
window/zone be small enough to reduce the scattered
background/excitation, lower the baseline Noise, increase
Signal/Noise ratio and improve detection sensitivity. It is
critical for the excitation fiber to be precisely positioned and
aligned such that a substantial portion of the light beam is
directed through the capillary wall at the separated sample
components in the capillary bore. Otherwise, the light can scatter
at the outside capillary wall/air interface and inside capillary
wall/buffer interface (Raman scattering), which can obscure or
corrupt the fluorescence emission intensity. The problem can be
multiplied if more than one fiber is used. Therefore, having one or
more excitation fiber positioned and aligned precisely with the
detection window is desirable.
Additionally, sample size and background noise pose additional
concerns in designing CE-based instruments. Only a relatively small
amount of DNA sample is being analyzed at any given time. As such,
the small sample emits fluorescence signals at levels that compete
with background noise. The background noise can come from the light
source, from Raman scattering, or from the materials of other
instrument components. The fluorescence signal can also scatter at
the wall interfaces. One or more lenses have been used to increase
detection sensitivity. However, a small misalignment of the
detection lens can have large effects on the detection sensitivity.
Accordingly, it is desirable for one or more detection lens
elements to be precisely positioned and aligned with the detection
window. Furthermore, having instrument components made from
materials that minimize background noise is desirable.
[0011] In the past, various techniques were developed for more
completely collecting the fluorescence emissions to improve signal
intensity and hence detection sensitivity. These techniques
involved additional moving and non-moving components that added to
the relative complexity and cost of the detection setup. Therefore,
it is desirable to have a means for CE analyses that is versatile
enough for use in a laboratory setting as well as being capable of
incorporation into a CE-based instrument capable of various
detection techniques. Additionally, this also calls for a means of
producing and assembling instruments at low cost.
SUMMARY OF THE INVENTION
[0012] The present invention provides an apparatus for precisely
aligning the optical detection components of a bio-separation
system. In a capillary electrophoresis (CE) system, for example,
the apparatus facilitates alignment of a capillary, one or more
excitation fibers, and one or more optical lens elements for
detection. In one aspect of the present invention, the apparatus is
capable of aligning the capillary relative to one or more
excitation fibers and relative to one or more optical lens
elements. The apparatus comprises an alignment block for aligning
the capillary, the fibers and the lens elements with respect to
each other and a support block for maintaining these components in
alignment. The alignment block includes a plurality of grooves for
aligning the one or more fibers with respect to a detection window
of the capillary. The support block can mate with the alignment
block to maintain the capillary and the one or more fibers within
the grooves. The apparatus also includes a lens seat on the
alignment block for optically aligning the optical axis of the lens
element with respect to the capillary axis and fiber(s) axis. By
supporting multiple excitation fibers and multiple lens elements,
the apparatus can be adapted for numerous CE detection schemes
(e.g., fluorescence or absorbance type detections).
[0013] In another aspect of the present invention, the apparatus
includes the alignment and the support blocks being identical
blocks capable of mating with each other for assembly into the
apparatus. Each block includes a plurality of grooves that form a
plurality of shafts for aligning the excitation fibers with respect
to the capillary when the blocks have mated, and a lens seat for
optically aligning the lens element with the capillary.
Furthermore, each block includes a locking mechanism having a male
part and a female part. The male part of one block can mate in a
press fit with the corresponding female part of the other block,
which provides easy assembly of the apparatus without fasteners.
Additionally, the locking mechanism provides a means for aligning
the two blocks with respect to each other.
[0014] In a further aspect of the present invention, an assembly of
a linear array of apparatuses is provided for incorporation into a
multi-capillary CE instrument. The assembly includes a bracket
capable of supporting a plurality of apparatuses in a linear array.
The assembly can further be incorporated in a cartridge for use in
a multi-capillary CE system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For a fuller understanding of the nature and advantages of
the invention, as well as the preferred mode of use, reference
should be made to the following detailed description read in
conjunction with the accompanying drawings. In the following
drawings, like reference numerals designate like or similar parts
throughout the drawings.
[0016] FIG. 1 is a schematic view of a capillary electrophoresis
system.
[0017] FIG. 2 is a schematic view of the excitation system.
[0018] FIG. 3 is a cross-sectional view of the detector probe.
[0019] FIG. 4 is a perspective view of the alignment apparatus in
accordance with one embodiment of the present invention.
[0020] FIG. 5 is a perspective view of the alignment block in
accordance with one embodiment of the present invention.
[0021] FIG. 6 is a cross-sectional view of the alignment apparatus
shown in FIG. 4, taken through line 6-6.
[0022] FIG. 7 is a cross-sectional view of the detector probe
inserted within the detector port.
[0023] FIG. 8 is a cross-sectional view of the alignment apparatus
shown in FIG. 2 taken along line 8-8.
[0024] FIG. 9A is a simplified drawing of a fluorescence detection
scheme employing two excitation fibers and two detection
lenses.
[0025] FIG. 9B is a simplified drawing of a fluorescence detection
scheme employing four excitation fibers and a single detection
lens.
[0026] FIG. 9C is a simplified drawing of an absorbance optical
detection scheme employing a light probe and the detection
probe.
[0027] FIG. 9D is a simplified drawing of a detection scheme
employing a surface mount LED light source and two detection
fibers.
[0028] FIG. 10 is a perspective view of a linear array support
bracket in accordance with one embodiment of the present
invention.
[0029] FIG. 11 is a perspective view of an assembly of a linear
array of alignment blocks.
[0030] FIG. 12 is a perspective view of an assembly of a linear
array of support blocks.
[0031] FIG. 13 is a perspective view of an assembly of a linear
array of alignment apparatuses.
[0032] FIG. 14 is a cross-sectional view of the assembly of the
linear array of alignment apparatuses shown in FIG. 13, taken
through line 14-14.
[0033] FIG. 15 is an exploded perspective view of a mid-section
body of a multi-capillary cartridge, the assembly of the linear
array of alignment blocks and the assembly of the linear array of
support blocks.
[0034] FIG. 16 is a perspective view of a multi-channel CE
instrument, which incorporates the alignment apparatus of the
present invention, in accordance with one embodiment of the present
invention.
[0035] FIG. 17 is a front perspective sectional view of the
cartridge.
[0036] FIG. 18 is a block diagram of the controller.
[0037] FIG. 19 is a perspective view of another embodiment of a
cartridge for use with the instrument shown in FIG. 16.
[0038] FIGS. 20 and 21 are front and rear perspective views of the
interface mechanism in accordance with one embodiment of the
present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] This invention is described below in reference to various
embodiments with reference to the figures. While this invention is
described in terms of the best mode for achieving this invention's
objectives, it will be appreciated by those skilled in the art that
variations may be accomplished in view of these teachings without
deviating from the spirit or scope of the invention.
[0040] The present invention is directed towards a novel apparatus
for precisely aligning components of a CE system, including a
capillary column, one or more excitation fibers, and one or more
optical lens elements. For the purpose of illustrating the
principles of the present invention and not by limitation, the
present invention is described by reference to embodiments directed
to capillary electrophoresis and radiation induced
fluorescence.
[0041] Referring to FIG. 1, a bio-separation system, and more
specifically a capillary electrophoresis (CE) system 1, is
schematically illustrated. The CE system 1 generally comprises a
capillary separation column 2 (e.g., 200-500 .mu.m O.D.), which
defines a micro-bore separation channel 3 (e.g., 20-250 .mu.m
I.D.). The capillary column 2 may be made of fused silica, glass,
polyimide, or other plastic/ceramic/glassy materials. The inside
walls of the capillary 2 (i.e., the walls of the separation channel
3) may be coated with a material that can build up an electrostatic
charge to facilitate electrophoresis and/or electrokinetic
migration of the sample components. The separation channel 3 may be
filled with a separation support medium, which may simply be a
running buffer, or a sieving gel matrix well known in the art. For
radiation induced fluorescence detection, the gel matrix includes a
known fluorophore, such as Ethidium Bromide.
[0042] One end of the capillary 2 is submerged in a reservoir 9 of
running buffer/gel 10, and the other end of the capillary 2 is
coupled to a sample vial 11. The capillary 2 is provided with a
detection window 12. The detection window 12 is a section of the
separation channel 3 wherein the Polyimide coating is pre-burned or
removed to define a transparent section of the separation channel
3. The detection window 12 can be located at an end section of the
capillary 2 near to the gel-reservoir 9. Radiation from a radiation
source 13 (e.g., LED or laser), which is part of a sample analysis
system, is carried through an excitation fiber 14 and is directed
from outside the capillary 2 through the detection window 12 at the
analytes. A radiation detector 15, also part of the sample analysis
system, is positioned outside the detection window 12. Electrodes
16 and 17 are coupled to the buffer reservoir 11 and gel reservoir
9 to complete the electrophoresis path.
[0043] For the sake of completeness, it is sufficient to briefly
mention the operation of the CE system 1. In operation, a prepared
biological sample (e.g., a DNA sample), direct from a Polymerase
Chain Reaction (PCR) machine is introduced into the far end of the
capillary 2 away from the detection window 12 by any of a number of
ways that is not part of the present invention (e.g.,
electrokinetic injection from a sample reservoir or physical
pressure injection using a syringe pump). The sample binds to the
fluorophore.
[0044] When a DC potential (e.g., 1-30 KV) is applied between
electrodes 16 and 17, the sample migrates under the applied
electric potential along the separation channel 3 (e.g. DNA that is
negatively charged travels through the sieving gel with an
integrated dye matrix/fluorophore toward a positive electrode as
shown in FIG. 1) and separates into bands of sample components. The
extent of separation and distance moved along the separation
channel 3 depends on a number of factors, such as migration
mobility of the sample components, the mass and size or length of
the sample components, and the separation support medium. The
driving forces in the separation channel 3 for the separation of
samples could be electrophoretic, pressure, or electro-osmotic flow
(EOF) means.
[0045] When the sample reaches a detection zone 27 within the
detection window 12, excitation radiation is directed via the
excitation fiber 14 at the detection zone 27. The sample components
fluoresce with intensities proportional to the concentrations of
the respective sample components (proportional to the amount of
fluorescent tag material). The radiation detector 15 detects the
intensities of the emitted fluorescence at a wavelength different
from that of the incident radiation. The detected emitted radiation
may be analyzed by known methods. The detection zone 27 is not
necessarily a well-defined zone with well-defined boundaries. This
is due to the nature of the sample, the incident radiation and the
radiation emission. The detection zone 27 is generally a zone
wherein radiation from the excitation fiber 14 is directed and from
where radiation emission from the radiated sample originates. For
an automated system, a controller 28 controls the operations of the
CE system 1.
[0046] The CE system 1 includes an excitation system 31 for
providing and directing radiation at the separated DNA fragments
within the detection zone 27. FIG. 2 is a schematic view of the
excitation system 31. The excitation system 31 includes the
radiation or light source 13, a coupling lens (e.g. micro-ball
lens) 32, and the excitation fiber 14.
[0047] The light source 13 can be a fluorescence excitation light
source such as an LED or a laser. The attractive features of LED's
as light sources are their low cost, small size, long lifetime,
good intensity and stability resulting in low noise, and the
possibility of direct electronic modulation of the intensity. The
LED's can be based on InGaN material technology (e.g., HLMP-CB15
and HLMP-CM15 from Agilent) with an average light output power of
2.5-3 mW. Different color LED's (e.g., blue or green LED's) could
be used as excitation sources for excitation of different
fluorophores (different applications). The light from the LED's can
be in wavelength ranges of 300-900 nm, and specifically at 524 nm.
Surface Mount (SMT) type LED's could also be used.
[0048] The excitation light source 13 can also be Laser Diodes
(semiconductor solid-state lasers) in the range of 400-800 nm.
Alternatively, they could be pulsed lasers (e.g., solid state
lasers, gas lasers, dye lasers, fiber lasers).
[0049] The excitation system 31 includes the micro-ball lens 32 and
the excitation fiber 14. The micro-ball lens 32 couples the light
from the radiation source 13 (i.e., the LED or laser) to enter the
excitation fiber 14. The excitation fiber 14 receives the light
from the lens 32 and directs the light to the detection zone 27.
The excitation fiber 14 can be a light transmitting optical fiber
(e.g., multi-mode silica or plastic 200 micron Core fibers, 0.22
N.A.). The Numerical Aperture (N.A.) of the excitation fiber 14
determines the amount of power density launched into the gel close
to the detection zone 27.
[0050] The CE system 1 includes the radiation detector 15 for
detecting the radiation/light from the sample in the detection zone
27. The radiation detector 15 can include a detector probe 41, such
as one referenced in U.S. patent application Ser. No. 10/060,052,
entitled "Optical Detection in A Multi-Channel Bio-Separation
System," filed on Jan. 28, 2002 (Attorney Docket No. 1031/209),
which is assigned to BioCal Technology, Inc., the assignee of the
present invention, and which is fully incorporated by reference
herein. FIG. 3 is a cross-sectional view of the detector probe
41.
[0051] The detector probe 41 includes a probe housing 43 for
housing a lens 44 and an emission collection fiber 45. The
florescence emissions from the separated components or analytes at
the detection zone 27 is collected through the lens 44, and
directed through the emission collection fiber 45 to a detector
(not shown). The capillary 2 may have transparent walls, or opaque
walls provided with a transparent window to direct emissions to the
lens 44. The lens 44 can be a collimation lens for collecting
emissions and can have a high collection angle property (e.g., a
sapphire micro-lens with index of refraction of n=1.76 from Swiss
Jewel Company Model # B2.00 that has a short focal distance with a
high numerical aperture (N.A.)). The lens 44 can also be an
emission coupling lens (e.g., a BK-7 or Sapphire micro-lens,
available from the Swiss Jewel Co.) for coupling the collimated
emission light produced by the collimation lens to the emission
collection fiber 45. The fluorescent light, which has a higher
wavelength (e.g., 570 to 630 nm) than the excitation light (500-550
nm), is then routed by the emission collection fiber 45 (e.g., a
large core optical fiber (370 .mu.m O.D., 0.22 NA fiber, but could
also be in ranges of: 100-1000 .mu.m O.D., 0.12-0.5 NA)) to the
detector (e.g., R5984 Hamamatsu photo-multiplier tube (PMT)) after
going through color separation (e.g., using 570-630 nm) long pass
or band pass emission filters. At the detector (PMT detector), the
emission signal is filtered by a single or multiple emission
filters (not shown) and can be read (detected) in a
time-multiplexed (time-staggered) scheme.
[0052] Referring to FIG. 1, the CE system 1 includes an alignment
apparatus or coupler 51 for positioning and aligning the excitation
fiber 14 of the excitation system 31 with respect to the detection
zone 27 of the capillary 2, and for optically coupling the
radiation detector 15 to the detection zone 27. FIG. 4 is a
perspective view of the alignment apparatus 51 in accordance with
one embodiment of the present invention. The alignment apparatus 51
includes an alignment block 52 and a support block 53.
[0053] FIG. 5 is a perspective view of the alignment block 52 in
accordance with one embodiment of the present invention, and FIG. 6
is a cross-sectional view of the alignment apparatus 51 shown in
FIG. 4 taken through line 6-6. The alignment block 52 includes an
outer face 57 and an opposing inner face 58. In the embodiment
shown in FIGS. 5 and 6, the outer and inner faces 57 and 58 have a
circular shape. However, the outer and inner faces 57 and 58 can
have other simple geometric shapes, such as a square shape. The
outer face 57 includes a plurality of grooves or channels, such as
grooves 59, 60 and 61, defined on the outer face 57.
[0054] The grooves 59, 60 and 61 facilitate positioning of the
capillary column 2 and the excitation fiber 14 in precise alignment
to each other. The grooves 59, 60 and 61 intersect each other at a
detection point 62 on the alignment block 52. The detection point
62 can be at any point on the outer face 57, such as at the center
of the outer face 57, as shown in FIG. 5. A groove can span from
one edge of the periphery of the outer face 57 to another edge,
such as groove 59, or can span from one edge of the periphery to
the detection point 62, such as grooves 60 and 61.
[0055] The grooves 59, 60 and 61 are sized and shaped to receive
either the capillary 2 or the excitation fiber 14. The capillary 2
or the excitation fiber 14 can be nested within the grooves 59, 60
and 61, and positioned such that the capillary 2 or the excitation
fiber 14 are precisely aligned with each other. More specifically,
the grooves 60 and 61 align the excitation fibers 14 relative to
the detection window 12. For example, the capillary 2 can be nested
in the groove 59 such that the detection window 12 is positioned
about the detection point 62. The alignment block 52 is capable of
supporting one or more excitation fibers 14. In the embodiment in
shown in FIG. 5, up to four excitation (or emission collection)
fibers 14 can be directed at the detection point 62 where the
detection window 12 is positioned. For example, a fiber can be
nested in groove 60 and another fiber in groove 61. The grooves 60
and 61 guide the excitation fibers 14 to the detection window 12
until the ends of the fibers 14 butt the outer diameter of the
capillary 2. The closer the fibers 14 are positioned to the
analytes in the detection zone 27, the more excitation energy is
directed towards the analytes and the stronger the emission signal.
The grooves allow for precise intersection of the capillary and
fibers centerlines. For example, a capillary having a 50 .mu.m I.D.
requires that the fiber centerline be located within 10 .mu.m of
the capillary centerline. The alignment block 52 can include a
beveled opening 66 at the outer end of the grooves 59, 60 and 61.
The beveled openings 66 allow the capillary 2 and the excitation
fiber 14 to be more easily inserted into the grooves 59, 60 and
61.
[0056] Referring to FIGS. 5 and 6, the alignment block 52 includes
an optical coupling aperture 70. The aperture 70 is a window or
opening through the inner and outer faces 58 and 57 at the
detection point 62. The aperture 70 allows for optical coupling
between the detection window 12 on the outer face side 57, and the
detection optics 44 of the radiation detector 15 on the inner face
side 58 and prevents excessive leakage of scattered excitation
light to be detected by the detection lens 44 and proportionally
controls/reduces the Noise.
[0057] Referring to FIG. 6, the inner face 58 of the alignment
block 52 interfaces with the radiation detector 15. The inner face
58 includes a lens seat 73 for positioning and aligning the
radiation detector lens 44 with respect to the aperture 70. The
lens seat 73 can be a conical lens seat (as shown in FIG. 6) or
alternatively any configuration capable of receiving the detector
lens 44, such as a spherical lens seat (not shown). The tip of the
conical lens seat 73 opens to the aperture 70 to provide a passage
for optically coupling the detector lens 44 with the detection
window 12 on the outer face side 57.
[0058] The alignment block 52 can include a radiation detector port
77. The port 77 is adapted to receive the radiation detector 15 and
to hold and align the detector optic within the lens seat 73. In
the embodiment shown in FIG. 6, the port 77 is adapted to hold the
detector probe 41 shown in FIG. 3. The port 77 is a barrel-shaped
shell defining a cavity 78. The diameter of the cavity 78 is sized
to receive the outer diameter of the probe housing 43. The
barrel-shaped port 77 has two opposing ends, wherein one end of the
port 77 is connected to the alignment block 52, with the inner face
58 facing the cavity 78, and the opposing end is provided as a port
opening 79.
[0059] FIG. 7 is a cross-sectional view of the detector probe 41
inserted within the detector port 77. The probe 41 is inserted
through the port opening 79 until the lens 44 is seated within the
lens seat 73. The port shell guides the lens 44 into position
within the lens seat 73 when the probe 41 is inserted within the
port 77. Once seated within the lens seat 73, the lens 44 is
precisely aligned with the detection zone 27. The lens 44 is
optically coupled to the detection zone 27 through the aperture 70.
The lens seat 73 positions and aligns the lens 44 within a
predetermined distance of the detection zone 27. For example, a
capillary column having a 50 .mu.m I.D. requires that the
centerline of the capillary and the optical axis of the lens must
intersect within 5 .mu.m of each other.
[0060] Referring back to FIG. 6, the alignment apparatus 51
includes the support block 53. The support block 53 supports the
capillary 2 and the excitation fibers 14 within their respective
grooves by engaging with the outer face 57 of the alignment block
52. The alignment block 52 includes a locking mechanism 85 for
mechanically coupling the alignment block 52 to the support block
53.
[0061] In the embodiment shown in FIG. 6, the support block 53 is
an identical alignment block. As shown in FIG. 6, each block 52 and
53 has the locking mechanism 85 having a pair of male and female
parts 88 and 89. The male part 88 is a pin and the female part 89
is a catch sized and shaped to receive the pin 88. FIG. 6 shows the
alignment block 52 engaged with the support block 53. To engage the
two blocks 52 and 53 together to form the alignment apparatus 51,
the pin 88 of the alignment block 52 is mated with the catch 89 of
the support block 53, while the pin 88 of the support block 53 is
mated with the catch 89 of the alignment block 52. The pin 88 and
the catch 89 facilitate alignment of the two blocks 52 and 53 for
mating. Since the two blocks 52 and 53 are identical, the female
part 89 of one block is sized and shaped to receive the male part
88 of the other block. The pin 88 and the catch 89 are a press fit
to provide a centering effect that removes the diameters of the pin
88 and the catch 89 from the tolerance stacks affecting the
position of the capillary 2, the fibers 14, and the lens 44. The
press fit of the pin 88 and the catch 89 also allows for assembly
of the alignment apparatus 51 without use of fasteners.
[0062] Since the alignment block 52 and the support block 53 mate
at a plane coincident with the axis of the capillary 2, the
alignment block 52 and the support structure block 53 can be
attached radially to the capillary 2, thus precluding the need to
string the alignment apparatus 51 onto the capillary 2. This allows
the alignment apparatus 51 to be attached to the capillary 2 after
end fittings have been fitted to the capillary 2.
[0063] FIG. 8 is a cross-sectional view of the alignment apparatus
51 shown in FIG. 2 taken along line 8-8. FIG. 8 shows the alignment
block 52 in engagement with the support block 53. Once the blocks
52 and 53 have mated, a groove of the alignment block 52 interfaces
with a corresponding groove of the support block 53 to form a shaft
into which the capillary 2 or the excitation fibers 14 can be
positioned into. The locking mechanism 85 allows the corresponding
grooves to precisely align to form the shaft. FIG. 8 shows, for
example, a shaft 92 for receiving the capillary 2. The capillary 2
is shown positioned within the channel 92. The plurality of shafts
are sized to receive the outer diameter of either the capillary 2
or the excitation fiber 14. The shafts facilitate positioning and
alignment of the detection window 12 of the capillary 2 and the
excitation fibers 14 with each other. Furthermore, FIG. 2 shows a
plurality of shaft openings 93 defined by the beveled openings 66
of the plurality of grooves 59, 60 and 61. The shaft openings 93
allow the capillary 2 and the excitation fibers 14 to be more
easily inserted into the shafts.
[0064] The support block can have any configuration which allows
support of the capillary and the excitation fibers within the
plurality of grooves. For example, the support block can be a
simple plate structure having a face capable of engaging with the
outer face of the alignment block (not shown). The alignment block
can also be provided with any locking mechanism that allows for
securely coupling the alignment block with the support block, such
as screwing or gluing the two blocks together.
[0065] The alignment apparatus 51 of the present invention is
capable of aligning multiple excitation fibers 14 relative to a
single capillary 2. Additionally, the alignment apparatus 51 of the
present invention is capable of optically coupling one or two
detection lenses 44 with a single capillary 2. In the embodiment of
the alignment apparatus 51 shown in FIG. 4 and in the embodiment of
the alignment block 52 shown in FIG. 5, the alignment apparatus 51
can support up to four excitation fibers 14 to one capillary 2, and
can couple up to two micro-ball lenses 44 with one capillary 2.
This arrangement allows for two emission detection lenses 44 to be
coupled from the two sides of the capillary 2 (180 degrees with
respect to each lens) to increase the emission collection light and
enhance the detection sensitivity. The same approach could also be
applied in the case of using two separate excitation fibers (for
two different excitation wavelengths) and detection of dual
wavelengths by two detection lenses from one capillary.
[0066] FIGS. 9A through 9D illustrate example detection schemes
that can be employed with the alignment apparatus 51 of the present
invention. FIG. 9A is a simplified drawing of a fluorescence
detection scheme employing two excitation fibers 98 and 99, and two
detection lenses 100 and 101. The capillary 2 can be nested in the
groove 59 with the detection window 12 positioned about the
detection point 62 (as shown in FIG. 5). The fiber 98 can be
located in the groove 60 and the fiber 99 can be located in a
groove 102. The fibers 98 and 99 deliver excitation light from the
radiation source 13 to the analytes within the detection window 12
of the capillary 2. The lens 100 can be located within the lens
seat 73 of the alignment block 52, and the lens 101 can be located
within the lens seat 73 of the support block 53. Both lenses 100
and 101 are optically coupled to the detection zone 27. The lenses
100 and 101 collimate the fluorescence signal from the detection
zone 27 and direct the signal to the PMT's. Use of the two lenses
100 and 101 permit a multi-color fluorescence optical detection
scheme.
[0067] FIG. 9B is a simplified drawing of a fluorescence detection
scheme employing four excitation fibers 98, 99, 105 and 106, and
the detection lens 100. The capillary 2 can be nested in the groove
59 with the detection window 12 positioned about the detection
point 62 (as shown in FIG. 5). The fiber 98 can be nested in the
groove 60, the fiber 99 can be nested in the groove 102, the fiber
105 can be nested in the groove 61 and the fiber 106 can be nested
in a groove 107. The fibers 98, 99, 105 and 106 deliver excitation
light from the radiation source 13 to the analytes within the
detection window 12 of the capillary 2. The lens 100 can be seated
within the lens seat 73 of the alignment block 52 or within the
lens seat 73 of the support block 53. The lens 100 is optically
coupled to the detection zone 27. The lens 100 collimates the
fluorescence signal from the detection zone 27 and directs the
signal to the PMT's via fibers.
[0068] The excitation system can be provided in a light probe (not
shown) for use in an absorbance optical detection scheme. The light
probe includes a light source, a light transmitting fiber, and a
micro-ball lens. Light from the light source (e.g., LED, laser, D2,
Xenon or Mercury lamps) is directed through the light transmitting
fiber to the micro-ball lens. The light probe can be inserted into
the port 77 of the alignment block 52, such that when the light
probe is inserted within the port 77, the micro-ball lens is seated
within the lens seat 73 of the alignment block 52. Once within the
lens seat 73, the micro-ball lens can direct the light to the
detection window 12 through the aperture 70.
[0069] FIG. 9C is a simplified drawing of an absorbance optical
detection scheme employing a light probe 112 and the detection
probe 41. The capillary 2 can be nested in the groove 59 with the
detection window 12 positioned about the detection point 62 (shown
in FIG. 5). Each of the light probe 112 and the detection probe 41
can be inserted in the port 77 of either the alignment block 52 or
the support block 53. A micro-ball lens 113 of the light probe 112
can be seated in the lens seat 73 of either the alignment block 52
or the support block 53. The lens 113 is optically coupled to the
detection zone 27 through the aperture 70. The lens 113 can direct
light from the light source to the detection window 12. The
micro-ball lens 44 of the detection probe 41, therefore, can be
seated in the other lens seat 73 of the alignment apparatus 51. The
lens 44 is optically coupled to the detection zone 27 through the
aperture 70. The lens 44 can detect the signal from the detection
zone 27 and can direct the signal to a detector (e.g., PMT).
[0070] FIG. 9D is a simplified drawing of a detection scheme
employing a surface mounted LED light source 116 and two detection
fibers 117 and 118. The capillary 2 can be nested in the groove 59
with the detection window 12 positioned about the detection point
62 (shown in FIG. 5). The surface mounted LED light source 116
provides excitation light that is directed to a coupling micro-ball
lens 119. The lens 119 is seated in the lens seat 73, such that the
lens 119 optically couples the light to the detection window 12
through the aperture 70. The fibers 117 and 118 can be nested in
the grooves 60 and 102, for example. The fibers 117 and 118 direct
signals from the detection zone 27 to a detector (e.g., PMT).
[0071] The alignment block 52 of the present invention can be
formed from any material that does not significantly fluoresce at
critical wavelengths, such as aluminum, stainless steel, copper,
platinum, Gold, silver, glass, ceramic, zinc, and non-fluorescing
plastics such as ESD. The materials thus minimize the background
noise, and therefore increase the detection sensitivity of the CE
system 1.
[0072] The simple geometric features of the alignment block 52 of
the present invention allow the alignment block 52 to be fabricated
by a variety of fabrication methods well known in the art, such as
die-casting, tooling and injection molding. Accordingly, the simple
geometric features allow the alignment block 52 to be producible at
a low cost.
[0073] The alignment apparatus 51 of the present invention also
allows for easy assembly of the apparatus 51. In the embodiment
shown in FIG. 4, the components of the alignment apparatus 51 is
comprised only of the two identical blocks 52 and 53. Each block 52
and 53 has the relatively simple locking mechanism 85. Assembly of
alignment apparatus 51 requires only to align the respective pins
88 and catches 89 of the two blocks 52 and 53 and to simply snap
the two identical blocks 52 and 53 together.
[0074] The relatively simple geometric features of the alignment
block 52 and the relative ease in assembling the alignment
apparatus 51 allow the alignment apparatus 51 to be employed in a
variety of environments. For example, the alignment apparatus 51
can be incorporated in a relatively mobile CE system for use in an
environment where use of a larger CE-instrument is not practical,
such as a laboratory setting. The mobile CE system can simply
include the alignment apparatus 51 for coupling and aligning a
single capillary with the excitation system 31 and radiation
detector 15. Multiple alignment apparatuses 51 can also be fitted
along the length of the capillary 2 for zone detection.
[0075] Additionally, a plurality of alignment apparatuses 51 (shown
in FIG. 4) can be provided in a linear array for integration into a
multi-capillary CE-instrument. FIG. 10 is a perspective view of a
linear array support bracket 122 in accordance with one embodiment
of the present invention. The support bracket 122 has an I-shaped
configuration having a plurality of holes 123 defined through a
middle flange 124. The plurality of holes 123 are sized and shaped
to receive the outer diameter of the alignment block 52 (shown in
FIG. 5).
[0076] FIG. 11 is a perspective view of an assembly 125 of a linear
array of alignment blocks 52. The assembly 125 includes the support
bracket 122 holding and positioning a plurality of alignment blocks
52 in a linear array. Each alignment block 52 is removeably
supported within one of the holes 123 of the support bracket 122.
The support bracket 122 allows for one or more alignment blocks 52
to be fitted and removed depending upon the particular requirements
of the application.
[0077] FIG. 12 is a perspective view of an assembly 126 of a linear
array of support blocks 53. The assembly 126 includes another
support bracket 127 for holding and positioning the plurality of
support blocks 53 in a linear array to provide the assembly 126 of
the liner array of support blocks 53. Each support block 53 is
removeably supported within one of the holes of the support bracket
127. The support bracket 127 allows for one or more alignment
blocks 53 to be fitted and removed depending upon the particular
requirements of the application.
[0078] FIG. 13 is a perspective view of an assembly 131 of a linear
array of alignment apparatuses 51. The assembly 131 includes the
assembly 125 (shown in FIG. 11) and the assembly 126 (shown in FIG.
12) in engagement with each other to form the linear array of
alignment apparatuses 51. FIG. 14 is a cross-sectional view of the
assembly 131 of the linear array of alignment apparatuses shown in
FIG. 13, taken through line 14-14. The assembly 131 can be
assembled by mating one of the plurality of alignment blocks 52 of
the assembly 125 with a corresponding support block 53 of the
assembly 126 (as detailed herein) until all of the plurality of
alignment blocks 52 are mated with their corresponding support
blocks 53. Each alignment apparatus in the array is capable of
providing optical shielding to minimize cross talking across other
alignment apparatuses 51 in the array.
[0079] The assembly 131 allows for multi-capillary CE. Each
alignment apparatus 51 of the assembly 131 can support one
capillary 2. Furthermore, each alignment apparatus 51 of the
assembly 131 can locate one or more excitation fibers 14 for the
capillary 2 and can optically couple one or two lenses to the
detection window 12 of the capillary 2.
[0080] The assembly 131 can be incorporated into a multi-capillary
CE-instrument. Reference is made to U.S. patent application Ser.
No. 10/060,052, entitled "Optical Detection In A Multi-Channel
Bio-Separation System," filed on Jan. 28, 2002 (Attorney Docket
No.: 1031/209), which is commonly assigned to BioCal Technology,
Inc., the assignee of the present invention, which is fully
incorporated by reference herein, and to U.S. patent application
Ser. No. 10/059,993 entitled "Multi-Channel Bio-Separation
Cartridge," filed on Jan. 28, 2002 (Attorney Docket No. 1031/208),
which is commonly assigned to BioCal Technology, Inc., the assignee
of the present invention, and which is fully incorporated by
reference herein.
[0081] FIG. 15 is an exploded perspective view of a mid-section
body of a multi-capillary cartridge 137, the assembly 125 of the
linear array of alignment blocks and the assembly 126 of the linear
array of support blocks. The assemblies 125 and 126 can be engaged
with each other at a section 138 of the mid-section body of the
multi-capillary cartridge 137. The assembly including the
mid-section body 137 incorporated with the assemblies 125 and 126
can be further incorporated with the multi-capillary CE-system (not
shown). The assembly 131 (shown in FIG. 13) allows for positioning
one or more capillaries in a linear array within a particular
design requirement, such as 9 mm or less (9 mm is the pitch
distance for a standard 96-well micro-titer plate).
[0082] In another embodiment of the present invention (not shown),
the plurality of alignment blocks and the plurality of support
blocks are integral or unitary with the linear array brackets.
[0083] FIG. 16 is a perspective view of a multi-channel CE
instrument 1200, which incorporates the alignment apparatus of the
present invention, in accordance with one embodiment of the present
invention. The fully automated DNA analysis instrument 1200 has a
base 1074, supporting a modular X-Z sample handling tray mechanism
1076, which moves two 96-well micro-titer plates 1070 and 1072 in
relation to the multi-capillary cartridge 1100, which can be
similar to the cartridge 137 (shown in FIG. 15) and can incorporate
similar structures, including the alignment apparatus of the
present invention.
[0084] The instrument 1200 incorporates the cartridge 1100. The
cartridge 1100 includes a twelve-channel fused silica capillary
array that is used for separation and detection of the samples as
part of a disposable and/or portable, interchangeable cartridge
assembly 1100. The multi-channel capillary array includes twelve
detection zones defined by micro-channels in the cartridge 1100.
The multi-channel cartridge 1100 shown in FIG. 16 holds up to 12
capillaries 1140, 12-16 cm long. The multi-channel cartridge 1100
is integrated with a top, outlet buffer reservoir 1124 common to
all capillaries 1140, which is directly coupled to a modular air
pressure pump 1078. The pressure pump 1078 provides the required
air pressure to fill-up all the 12-capillaries 1140 with the
sieving gel contained in the reservoir 1124. Depending on the
viscosity of the gel, pressures of up to 40 PSI may be applied to
the capillaries 1140 through the gel-filled reservoir 1124. The
cartridge gel-reservoir 1124 is equipped with built in common
electrode (anode; not shown) for all 12-capillaries 1140, which is
automatically connected to a high voltage power supply 1080 for
electrophoresis when installed inside the instrument 1200.
Injection of the samples is achieved by electrokinetic methods. The
high voltage power supply 1080 is used to deliver 0-to-20 KV of
electrical field to the gel-filled capillaries for the
electrokinetic injection and separations of DNA fragments. Each of
the 12-LED's broad band light energy (FWHM=47 nm) is relayed by
individual light transmitting optical fibers (multi-mode silica or
plastic 200 micron Core fibers, 0.22 N.A.) to each of the
capillary's detection zone inside the cartridge 1100 for the
excitation of the separated DNA fragments. A power supply 1066
provides DC power to the CE instrument 1200.
[0085] FIG. 17 is a front perspective sectional view of the
cartridge 1100. The structure of the lower body of cartridge 1100
provides the optical alignment means or coupling of LEDs 2184,
micro-ball lenses 2182, and excitation fibers 2116 inside the
cartridge 1100. The excitation light from the LEDs 2184 is directed
through the excitation fibers 2116 to detection zones 2155 of the
capillaries. An array of detection probes 2170 is connected at the
rear side of the cartridge 1100.
[0086] FIG. 19 is a perspective view of another embodiment of a
cartridge 3000 for use with the instrument 1200 shown in FIG. 16.
This embodiment of the cartridge 3000 is designed for use with an
interface mechanism 3100 of the instrument 1200. FIGS. 20 and 21
are front and rear perspective views of the interface mechanism
3100 in accordance with one embodiment of the present invention.
The interface mechanism 3100 provides for precise and repeatable
mechanical positioning of the cartridge 3000 with an array of
detector probes 3200. The interface mechanism 3100 can drive the
array of detector probes 3200 to interface with the cartridge
3000.
[0087] The instrument 1200 includes a controller 1300. FIG. 18 is a
block diagram of the controller 1300. The controller 1300, which
comprises a CPU 1210, an A/D converter 1212 for converting
detection signals from the PMT to corresponding digital signals,
and an I/O interface 1214 for transferring and receiving signals to
and from respective parts of the CE instrument 1200 by instructions
from the CPU 1210. The I/O interface 1214 is coupled with a
temperature controller 1065, which controls the high-voltage power
supply for sample injection and electrophoresis functions of the CE
instrument 1200. The CPU 1210 may be further coupled to an external
personal computer 1218, which in turn performs data processing or
additional control function for the CE instrument 1200. The CPU
1210 and/or the PC 1218 may be programmed with control functions
dictated by LabVIEW.TM. software available from National
Instruments Corporation, to control various features and functions
of the automated multi-channel DNA instrument 1200.
* * * * *